Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase.

All IgG-type antibodies are N-glycosylated in their Fc part at Asn-297. Typically, a fucose residue is attached to the first N-acetylglucosamine of these complex-type N-glycans. Antibodies lacking core fucosylation show a significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and an increased efficacy of anti-tumor activity. In cases where the clinical efficacy of an antibody is to some extent mediated by its ADCC effector function, afucosylated N-glycans could help to reduce dose requirement and save manufacturing costs. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate here that heterologous expression of the prokaryotic enzyme GDP-6-deoxy-d-lyxo-4-hexulose reductase within the cytosol can efficiently deflect the fucose de novo pathway. Antibody-producing CHO cells that were modified in this way secrete antibodies lacking core fucose as demonstrated by MALDI-TOF mass spectrometry and HPAEC-PAD monosaccharide analysis. Engineering of the fucose de novo pathway has led to the construction of IgGs with a strongly enhanced ADCC effector function. The method described here should have broad practical applicability for the development of next-generation therapeutic antibodies.

Immunological substance testing on human lymphatic micro-organoids in vitro.

Pharmaceutical drugs and compounds used for consumer products may bear the risk of unexpected immuno-toxicological side effects, such as sensitization, allergy, anaphylaxis or immunogenicity. Modern biopharmaceuticals with high potency and target specificity, like antibodies and cytokines need to be tested for their therapeutical doses, their exposition regimens and their immune functionality prior to first-in-man applications. For the latter, existing in vitro tests and animal models do not sufficiently reflect the complexity and specificity of the human immune system. Even novel humanised animal models have limitations in their systemic reactions. Monolayer or suspended cell culture possesses neither tissue functionality nor organ physiology, and also cannot be used for long term culture and experiments. In contrast, solid tissue biopsies, e.g. tonsil preparations of tonsillitis patients typically show inflammatory artefacts and degrade in long term culture due to preparation-induced damage. The construction of tissue-like structures in vitro, so-called "micro-organoids", can overcome these limitations. Key structures of secondary lymphatic organs, e.g. lymph nodes or the spleen are the primary lymphatic follicles and germinal centres, in particular during the "activated state" of an inflammation or infection. To remodel lymphatic follicles, functional and structural cells, e.g. lymphoid cells derived from peripheral blood mononuclear cells (PBMCs) and stromal cells need to be combined with biogenic or artificial matrices and scaffolds to produce a suitable 3D tissue-mimicking environment. Therefore, a unique human lymph node model (HuALN) was designed to operate over several weeks, and allow long term and repeated drug exposure to induce and monitor both cellular and humoral immune responses. Cellular immunity is monitored, for example, by cytokine release patterns; humoral immunity is analysed, for example, by B cell activation, plasma cell formation and antibody secretion profiles (IgM and IgG). Moreover, cellular composition and micro-organoid formation are analysed by flow cytometry, histology and in situ imaging.

A human lymph node in vitro--challenges and progress.

Extracorporeal human lymphatic organs are expected to be excellent tools in the study of human molecular and cellular bases of the immunologic balance and tissue harmony. A rational approach and process to design a device and a procedure to recreate the human lymph node environment in vitro is described with emphasis on T-cell activation. Based on this approach, a bioreactor and a process supporting self-assembly of human lymphatic tissues due to proper emulation of human architecture and homeostasis could be developed.